*2.9. Fourier Transform Infrared Spectroscopy (FTIR) Analysis*

*2.9. Fourier Transform Infrared Spectroscopy (FTIR) Analysis* FTIR analysis provides information about the interactions among functional groups of film constituents. The FTIR pattern of the film samples based on pectin and collagen containing different concentrations of MOEO is shown in Figure 5. The 3300 cm−1 region band peaks reflected OH stretching bonds. Inter-molecular or intramolecular hydrogen bonds express the stretching vibration of OH [13]. The O=C=O stretching vibrations were responsible for the peak at the 2389 cm−1 region. The active films containing MOEO exhibited bands in those areas, suggesting that MOEO was linked to the biopolymers comprising film matrix, as depicted in Figure 5 [27]. The band at 2274 cm−1 was linked to the N=C=O isocyanate group, whereas the band at 1170–1220 cm−1 was linked to the C–O stretching vibrations. The stretching vibrations of –OH and hydrogen bonding among the hydroxyl groups of the film-forming components can be indicated by a broad peak observed in the 3600–3200 cm−1 range [28]. Films loaded with MOEO showed an increase in the peak intensities of (–OH) stretching as compared to the control film sample, which indicates the formation of new hydrogen bonds with the addition of oil in the film matrix. Furthermore, alterations in the FTIR spectrum were noted in oil-loaded samples, resulting FTIR analysis provides information about the interactions among functional groups of film constituents. The FTIR pattern of the film samples based on pectin and collagen containing different concentrations of MOEO is shown in Figure 5. The 3300 cm−<sup>1</sup> region band peaks reflected OH stretching bonds. Inter-molecular or intramolecular hydrogen bonds express the stretching vibration of OH [13]. The O=C=O stretching vibrations were responsible for the peak at the 2389 cm−<sup>1</sup> region. The active films containing MOEO exhibited bands in those areas, suggesting that MOEO was linked to the biopolymers comprising film matrix, as depicted in Figure 5 [27]. The band at 2274 cm−<sup>1</sup> was linked to the N=C=O isocyanate group, whereas the band at 1170–1220 cm−<sup>1</sup> was linked to the C–O stretching vibrations. The stretching vibrations of –OH and hydrogen bonding among the hydroxyl groups of the film-forming components can be indicated by a broad peak observed in the 3600–3200 cm−<sup>1</sup> range [28]. Films loaded with MOEO showed an increase in the peak intensities of (–OH) stretching as compared to the control film sample, which indicates the formation of new hydrogen bonds with the addition of oil in the film matrix. Furthermore, alterations in the FTIR spectrum were noted in oil-loaded samples, resulting in the appearance of new characteristic peaks (Figure 5). In general, the FTIR examination demonstrated the interaction among the functional groups of the film-forming constituents.

in the appearance of new characteristic peaks (Figure 5). In general, the FTIR examination demonstrated the interaction among the functional groups of the film-forming constitu-

ents.

**Figure 5.** Fourier transform infrared patterns of B (control), PC20 (containing 0.1% MOEO), PC30 (containing 0.15% of MOEO), and PC40 (containing 0.2% MOEO). **Figure 5.** Fourier transform infrared patterns of B (control), PC20 (containing 0.1% MOEO), PC30 (containing 0.15% of MOEO), and PC40 (containing 0.2% MOEO).

#### *2.10. Antioxidant Activities of the Hydrogel-Based Films 2.10. Antioxidant Activities of the Hydrogel-Based Films*

Edible films that contain essential oils or plant extracts exhibit promising antioxidant characteristics and offer a natural substitute for synthetic antioxidants. Additionally, these films enhance the sensory attributes, life expectancy, and overall quality of food products. The prepared hydrogel-based films containing MOEO were assessed for their antioxidant properties by using DPPH and ABTS assays. The results of the present investigation are shown in Figure 6, which indicate a rise in the DPPH and ABTS radical scavenging activity of the hydrogel-based films when loaded with MOEO. The highest DPPH and ABTS radical scavenging activity was shown by the PC40 film sample loaded with the highest concentration of MOEO; however, the lowest antioxidant activity was observed in the control film sample. The DPPH and ABTS radical scavenging activity in the film samples was enhanced with the addition of MOEO, increasing from 18.56 to 59.03% and from 64.31 to 73.75%, correspondingly. The improvement observed in the antioxidant activity of the films may be attributed to the existence of phenolic compounds in the MOEO. Edible films that contain essential oils or plant extracts exhibit promising antioxidant characteristics and offer a natural substitute for synthetic antioxidants. Additionally, these films enhance the sensory attributes, life expectancy, and overall quality of food products. The prepared hydrogel-based films containing MOEO were assessed for their antioxidant properties by using DPPH and ABTS assays. The results of the present investigation are shown in Figure 6, which indicate a rise in the DPPH and ABTS radical scavenging activity of the hydrogel-based films when loaded with MOEO. The highest DPPH and ABTS radical scavenging activity was shown by the PC40 film sample loaded with the highest concentration of MOEO; however, the lowest antioxidant activity was observed in the control film sample. The DPPH and ABTS radical scavenging activity in the film samples was enhanced with the addition of MOEO, increasing from 18.56 to 59.03% and from 64.31 to 73.75%, correspondingly. The improvement observed in the antioxidant activity of the films may be attributed to the existence of phenolic compounds in the MOEO.

Sani et al. [14] also reported similar results in which the sodium caseinate-based edible films possessed higher antioxidant activity when incorporated with a combination of zinc oxide nanoparticles (ZnONPs) and microcapsules of MOEO. Furthermore, many researchers have shown an increase in the antioxidant potency of edible films upon the incorporation of essential oils [28–30]. Sani et al. [14] also reported similar results in which the sodium caseinate-based edible films possessed higher antioxidant activity when incorporated with a combination of zinc oxide nanoparticles (ZnONPs) and microcapsules of MOEO. Furthermore, many researchers have shown an increase in the antioxidant potency of edible films upon the incorporation of essential oils [28–30].

#### **3. Conclusions 3. Conclusions**

The *Melissa officinalis* essential oil (MOEO) appears as an interesting ingredient for the formulation of pectin and collagen composite-based hydrogel films. MOEO-loaded films showed more elasticity and thickness; however, the tensile strength and transparency were lower. The addition of the highest concentration (0.2%) of MOEO improved the barrier and antioxidant properties of the hydrogel-based films. The findings of the present investigation indicate that composite hydrogel films composed of pectin and collagen loaded with MOEO exhibit promising prospects as a viable food packaging material. However, more research is needed to enhance the mechanical and optical properties of The *Melissa officinalis* essential oil (MOEO) appears as an interesting ingredient for the formulation of pectin and collagen composite-based hydrogel films. MOEO-loaded films showed more elasticity and thickness; however, the tensile strength and transparency were lower. The addition of the highest concentration (0.2%) of MOEO improved the barrier and antioxidant properties of the hydrogel-based films. The findings of the present investigation indicate that composite hydrogel films composed of pectin and collagen loaded with MOEO exhibit promising prospects as a viable food packaging material. However, more research is needed to enhance the mechanical and optical properties of the prepared films.

### the prepared films. **4. Materials and Methods**

#### **4. Materials and Methods** *4.1. Material Procurement*

*4.1. Material Procurement* Biopolymers including pectin and collagen with a purity of 95% were purchased from SRL Pvt. Ltd., Mumbai, India. The plasticizer (glycerol) was obtained from BDH Laboratory located in London, England. The Melissa oil used in this study was sourced from Nature Natural, a company based in Ghaziabad, India, and was identified by the batch Biopolymers including pectin and collagen with a purity of 95% were purchased from SRL Pvt. Ltd., Mumbai, India. The plasticizer (glycerol) was obtained from BDH Laboratory located in London, England. The Melissa oil used in this study was sourced from Nature Natural, a company based in Ghaziabad, India, and was identified by the batch number NNIMEEO/154/0821.

#### number NNIMEEO/154/0821. *4.2. Film Preparation*

analysis.

*4.2. Film Preparation* The film-forming solutions of pectin (1.5% *w*/*v*) and collagen (1% *w*/*v*) were prepared separately by using distilled water. After the complete dissolution of polymers in distilled water, both solutions were blended. The solution was distributed in four labeled beakers (B, PC-20, PC-30, and PC-40), and concentrations of Tween 80, MOEO, and glycerol were added in the film-forming solutions, as shown in Table 4. The resultant solutions were The film-forming solutions of pectin (1.5% *w*/*v*) and collagen (1% *w*/*v*) were prepared separately by using distilled water. After the complete dissolution of polymers in distilled water, both solutions were blended. The solution was distributed in four labeled beakers (B, PC-20, PC-30, and PC-40), and concentrations of Tween 80, MOEO, and glycerol were added in the film-forming solutions, as shown in Table 4. The resultant solutions were eventually transferred into the respective Petri dishes and then subjected to drying at suitable room temperature conditions. Finally, the films were peeled and stored for further analysis.

eventually transferred into the respective Petri dishes and then subjected to drying at suitable room temperature conditions. Finally, the films were peeled and stored for further


**Table 4.** The composition of the film-forming solution for different samples.
